The development of hybrid plant breeding has made possible considerable advances in quality and quantity of crops produced. Increased yield and the combination of desirable characteristics, such as resistance to disease and insects, heat and drought tolerance, and variations in plant composition are all possible, in part, due to hybridization procedures. Hybridization procedures rely on the contribution of pollen from a male parent plant to a female parent plant to produce the resulting hybrid.
Plants may self-pollinate if pollen from one flower is transferred to the same or another flower of the same plant. Plants may cross-pollinate if the pollen originates in a flower from a different plant. Maize plants (Zea mays) may be bred by both self-pollination and cross-pollination techniques. Maize plants have male flowers, which are located on the tassel, and female flowers, which are located on the ear of the same plant. Natural pollination in maize occurs when pollen from the tassels reaches the silks that are found at the tops of the incipient ears. The development of maize hybrids relies upon male sterility systems.
The development of maize hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection are two breeding methods used to develop inbred lines from populations. Breeding programs combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. A hybrid maize variety is the cross of two such inbred lines, each of which may have one or more desirable characteristics absent in one, or complementing the other. The new inbred plants are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which are desirable. The hybrid progeny from the first generation are designated F1. In the development of hybrids, only the F1 hybrids are sought. The F1 hybrid is typically more vigorous than its inbred parents. This hybrid vigor, termed heterosis, typically leads to, for example, increased vegetative growth and increased yield.
Hybrid maize seed can be produced by a male sterility system incorporating manual detasseling. To produce hybrid seed, the male tassel is removed from the growing female inbred parent, which can be planted in various alternating row patterns with the male inbred parent. Consequently, providing that there is sufficient isolation from foreign maize pollen, the ears of the female inbred will be fertilized only with pollen from the male inbred. The resulting seed is hybrid F1 seed.
Manual detasseling is labor-intensive and costly. Manual detasseling is also often ineffective, for example, because environmental variation in plant development can result in plants tasseling after manual detasseling of the female parent plant is completed, or because a detasseler might not completely remove the tassel of a female inbred plant. If detasseling is ineffective, the female plant will successfully shed pollen and some female plants will be self-pollinated. This will result in seed of the female inbred being harvested along with the hybrid seed which is normally produced. Female inbred see is not as productive as F1 seed. In addition, the presence of female inbred seed can represent a germplasm security risk for the producer of the hybrid seed.
A female inbred plant can also be mechanically detasseled by a machine. Mechanical detasseling is approximately as reliable as hand detasseling, but is faster and less expensive. However, most detasseling machines produces more damage to the plants than hand detasseling. Thus, no form of detasseling is presently entirely satisfactory.
Genetic male sterility is an alternative method that may be used in hybrid seed production. The laborious detasseling process can be avoided in some genotypes by using cytoplasmic male-sterile (CMS) inbred plants. In the absence of a fertility restorer gene, plants of a CMS inbred are male sterile as a result of factors resulting from the cytoplasmic, as opposed to the nuclear genome. Therefore, the characteristic of male sterility is inherited exclusively through the female parent in maize plants, since only the female provides cytoplasm to the fertilized seed. CMS plants are fertilized with pollen from another inbred that is not male-sterile. Pollen from the second inbred may or may not contribute genes that make the hybrid plants male-fertile. Usually seed from detasseled normal maize and CMS produced seed of the same hybrid must be blended to insure that adequate pollen loads are available for fertilization when the hybrid plants are grown and to ensure cytoplasmic diversity.
Drawbacks to CMS as a system for the production of hybrid seed include the association of specific variants to CMS with susceptibility to certain crop diseases. See, e.g., Beckett (1971) Crop Science 11:724-6. This problem has specifically discouraged the use of the CMS-T variant in the production of hybrid maize seed, and has had a negative impact on the use of CMS in maize in general.
Cytoplasmic male sterility (CMS) is the maternally inherited inability to produce functional pollen. More than 40 sources of CMS have been found and classified into three major groups by differential fertility restoration reactions in maize. These groups are designated as CMS-T (Texas), CMS-S (USDA) and CMS-C (Charrua). Beckett (1971). In the CMS-T group, two dominant genes, Rf1 and Rf2, which are located on chromosomes 3 and 9, respectively, are required for the restoration of pollen fertility. Duvick (1965) Adv. Genetics 13:1-56. The S-cytoplasm is restored by a single gene, Rf3, which has been mapped on chromosome 2. Laughnan and Gabay (1978) “Nuclear and cytoplasmic mutations to fertility in S male-sterile maize,” in Maize Breeding and Genetics, pp. 427-446.
Compared to CMS-T and CMS-S, the fertility restoration of CMS-C has been found to be very complex in previous analyses. Duvick (1972), “Potential usefulness of new cytoplasmic male sterile and sterility system,” in Proceeding of the 27th annual corn and sorghum research conference, pp. 197-201, found that full restoration of fertility in CMS-C is controlled by a dominant allele of Rf4 gene. Khey-Pour et al. (1981) also found this gene to be sufficient for CMS-C restoration. However, Josephson et al. (1978), “Genetics and inheritance of fertility restoration of male sterile cytoplasms in corn,” in Proceedings of the 33rd corn and sorghum research conference 7:13, proposed that full restoration of fertility in CMS-C was conditioned by the complementary action of the dominant alleles of two genes, Rf4 and Rf5, which have since been mapped to chromosomes 8 and 5, respectively. Sisco (1991) Crop Sci. 31:1263-6. Meanwhile, Chen et al. (1979) Acta Agronom. Sin. 5(4):21-28, considered that two dominant restorer genes in CMS-C had duplicate functions. Further complicating the system, Vidakovic (1988), Maydica 33:51-65, demonstrated the existence of three dominant and complementary genes for full restoration of fertility in CMS-C, adding the gene, Rf6. Vidakovic et al., (1997a) Maize Genet. Coop. News Lett. 71:10; (1997b) Maydica 42:313-6, later reported these complementary genes, Rf4, Rf5, and Rf6, were indeed not the sole genetic systems for fertility restoration in CMS-C of maize. Thus, the fertility restoration mechanisms of CMS-C remain unresolved. As a result, it is difficult to select restorer lines for some genotypic sterile lines.
Molecular markers are particularly useful for accelerating the process of introducing a gene or quantitative trait loci (QTL) into an elite cultivar or breeding line via backcrossing. Markers linked to the gene can be used to select plants possessing the desired trait, and markers throughout the genome can be used to select plants that are genetically similar to the recurrent parent (Young and Tanksley (1989) Theor. Appl. Genet. 77:95-101; Hospital et al. (1992) Genetics 132:1199-210).
Most of the plant fertility restorer genes have been cloned via a map-based cloning strategy. To date, nine Rf genes have been isolated from several plant species including maize (Zea Mays L.) (Cui et al. (1996) Science 272:1334-6; Liu et al. (2001) Plant Cell 13:1063-78), Petunia (Petunia hybrida) (Bentolila et al. (2002) Proc. Natl. Acad. Sci. USA 99:10887-92, radish (Raphanus sativus L.) (Brown et al. (2003) Plant J. 35:262-72; Desloire et al. (2003) EMBO Rep. 4:1-7; Koizuka et al. (2003) Plant J. 34:407-15), sorghum (Sorghum bicolor L.) (Klein et al. (2005) Theor. Appl. Genet. 111:994-1012), rice (Oryza sativa L.) (Kazama and Toriyama (2003) FEBS Lett. 544:99-102; Akagi et al. (2004) Theor. Appl. Genet. 108:1449-57; Komori et al. (2004) Plant J. 37:315-25; Wang et al. (2006) Plant Cell 18:676-87; and Fujii and Toriyama (2009) Proc. Natl. Acad. Sci. USA 106(23):9513-8), and monkey flower (Mimulus guttatus) (Barr and Fishman (2010) Genetics 184:455-65).
All of the identified restorer genes, except for Rf2 in maize and Rf17 in rice, encode different pentatricopeptide repeat (PPR) proteins. Plant genomes encode several hundred PPR proteins with many of them involved in regulating organelle gene expression. Lurin et al. (2004) Plant Cell 16:2089-103; and Schmitz-Linneweber and Small (2008) Trends Plant Sci. 12:663-70. A PPR protein contains 2 to 27 repeats of 35 amino acids, called PPR motifs. Small and Peeters, (2000) Trends Biochem. Sci. 25(2):46-7. PPR proteins are predicted to bind to RNA (Delannoy et al. (2007) Biochemical Society Transactions 35:1643-7), and many PPR proteins are targeted to mitochondria where the CMS-associated genes and products are located. Lurin et al. (2004), supra. Evidence suggest that PPR proteins bind directly to CMS transcripts. Akagi et al. (2004), supra; Gillman et al. (2007) Plant J. 49:217-27; and Kazama et al. (2008) Plant J. 55:619-28. Rf proteins reduce the expression of CMS-associated transcripts by changing their processing patterns (Kazama & Toriyama (2003), supra), decreasing RNA stability (Wang et al. (2006), supra; and Ohta et al. (2010) Plant Cell Rep. 29:359-69), or preventing them from being translated (Kazama et al. (2008), supra).
Additional information regarding restorer of fertility genes from maize, rice, petunia, and radish may be found in U.S. Patent Application Ser. No. US2006/0253931, and in U.S. Pat. Nos. 5,981,833; 5,624,842; 4,569,152; 6,951,970; 6,392,127; 7,612,251; 7,314,971; 7,017,375; 7,164,058; and 5,644,066.